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Oligonucleotide Synthesis
About Oligonucleotide Synthesis is the process of synthesizing fragments of DNA or RNA by methodically adding desired nucleotides. It is useful in the laboratories because it can be used to create DNA primers for procedures such as PCR replication, which is useful for DNA amplification. The idea of creating custom sequences easily and and cost efficient has changed the industry. Although oligonucleotide synthesis is a process that only creates strands around 15 to 20 base pairs in length, there have been developments in manufacturing that can construct long oligonucleotides for drug molecules. The method used in oligonucleotide synthesis is called "solid-phase," where the synthesis of the strand is supported by "solid supports" called resins. These resins are insoluble and essentially hold the oligonucleotide in place throughout the construction of the strand. As biological strands of DNA are synthesized from 5' to 3', oligonucleotide synthesis uses the opposite method, 3' to 5'. The 3' end of the strand is attached to the supports and is followed by a series of steps that will be discussed further. ] Method Development Oligonucleotide synthesis is typically done using the Phosphoramidite synthetic method. The idea of this method was created by McBride and Caruthers in 1983 where they used the 3' end of the first monomer to bind to the solid supports. The solid supports used are generally Polystyrene or Controlled Pore Glass (CPG). CPG is very effective because it has allowed for the synthesis of larger oligonucleotides. As the typical length of a synthesized oligonucleotide is 15-20 base pairs, CPG has changed the industry by having supports with pore sizes from 500 angstroms (Å) to 2000 Å. A 2000 Å CPG can create strands that are more than 100 base pairs in length. This synthesis method is the usage of DNA Phosphoramidite monomers where one nucleotide is added per cycle. A Phosphoramidite is a nucleotide that has protection groups (Trityl, hydroxyl, and phosphate) that protect the strand from unwanted additions throughout synthesis. The addition of nucleotides occurs from 3' to 5' (as previously stated) and follows four main steps: Deprotection, coupling, capping, and stabilization. Steps of Synthesis ] Deprotection Deprotection is also known as de-blocking or detritylation because the 5' DMT (4,4'-dimethoxytrityl) that protects the 5' hydroxyl (5'-OH) is removed by Trichloroacetic acid (TCA). Once this occurs there is an exposed hydroxyl group left behind. This is the addition site, hence the 3' to 5' direction in this synthesis process. Coupling Coupling is also known as base condensation, where the reactive 5'-OH from the deprotection step is ready to react with the next base monomer (phosphoramidite). Adding tetrazole (a weak acid) activates the phosphoramidite. At this point in the process the reactive 5'-OH and the reactive phosphorus atom form an unstable phosphite linkage. Capping Capping is the process that "caps" all of the free reactive groups. Sometimes coupling failures occur in which the oligonucleotide has a reactive 5'-OH group left unreacted. If this hydroxyl is not capped, it can, and will, react with other nucleotides in future cycles. This is bad because the sequence will lose a base in the desired sequence, resulting in a deletion. Adding acetic anhydride and N-methylimidazole acetylates alcohols (-OH groups) and permanently inhibits their ability to be reactive in subsequent cycles. Stabilization The stabilization step is also referred to as the oxidation step. This is the process of stabilizing the phosphate linkage between the oligonucleotide and the recently added base. Making the phosphite group stabile requires the addition of iodine, water, tetrahydrofuran, and pyridine, which oxidizes the phosphite to a phosphate. Repeat Repeating this cycle is done until the desired bases have been added to the nucleotide. Finalizing the Process Once the desired oligonucleotide is synthesized, the 3' end must be released from the solid supports and the protecting groups need to be removed, such as the 5'-DMT. Using a mixture of ammonia and methylamine allows this final process to occur. The result is exposed hydroxyl groups on the 3' and 5' ends which means that it can be used as a functional single strand of DNA. Usage in Genetics and Genomics Antisense Oligonucleotides are useful for therapeutic agents regarding disease. These specific oligonucleotides are used to bind mRNA which can effectively block ribosomal activity and stop the synthesis of a protein. Typically, these antisense oligonucleotides are attached to a targeting device to reach target cells. Research has been conducted for Hepatitis C, where the antisense oligonucleotide targets microRNA (miRNA) to suppress viral replication within the liver. References 1. Custom Oligonucleotide Synthesis (Davidson University) 2. Solid-Phase Oligonucleotide Synthesis (ATDBio) 3. Oligonucleotide Synthesis (Exiqon) 4. Chemical Synthesis and Purification of Oligonucleotides (Integrated DNA Technologies) 5. Synthesis of Oligonucleotides (Biomers.net) 6. Antisense Oligonucleotides (Kimball's Biology Pages)